Use of antimicrobial proteins and peptides for the treatment of otitis media and paranasal sinusitis

ABSTRACT

The pharmaceutical composition and a method of treatment of infectious diseases, such as otitis media, paranasal sinusitis, labyrinthitis and meningitis are described. The composition comprises EP2E or homologues thereof.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/971,559filed Oct. 22, 2004, which is a continuation-in-part of U.S. applicationSer. No. 10/819,714 filed Apr. 6, 2004, which is a continuation of U.S.application Ser. No. 09/998,547 filed Nov. 27, 2001, now U.S. Pat. No.6,716,813, which claims the benefit of U.S. Provisional Application No.60/253,492 filed Nov. 28, 2000, all of which are expressly incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to the use of human beta defensins,lysozyme, and lactoferrin as a new class of non-antibioticantimicrobials. More specifically, the invention relates to the use ofthese antimicrobials for the treatment of otitis media and paranasalsinusitis.

2. Description of the Related Art

The rapid worldwide increase in antibiotic resistance among pathogenshas given rise to an urgent need to develop new and innovativenon-antibiotic approaches to prevent and manage disease. Otitis media(OM) and sinusitis are two very common infections which are difficult totreat for a number of reasons, including antibiotic resistance. OM isthe most prevalent infectious disease affecting young children, and themajor cause of conductive hearing loss among this group. OM is also theleading indication for antibiotic therapy. OM results in 31 millionannual visits to physicians' offices and is estimated to have a yearlycost exceeding $5 billion.

OM occurs along a continuum. For example, OM with effusion characterizedby fluid pathology can lead to chronic OM plus chronic mastoiditis,characterized by the presence of intractable tissue pathology such ascholesteatoma, cholesterol granuloma or granulation tissue. Theliterature defines chronic OM as having a tympanic membrane perforationand otorrhea. Amongst many other sequelae, which can result from thecontinuum, an important common one is chronic silent OM. This overlookedentity which includes pathology beneath an intact tympanic membrane iscommonly seen in patients. Labyrinthitis is the most frequentcomplication of chronic OM.

Most cases of OM are caused by one of three major pathogens,Streptococcus pneumoniae (S. pneumoniae) (30-40%), non-typeableHaemophilus influenzae (NTHi) (30%) and Moraxella catarrhalis (M.catarrhalis) (20%). These are the same pathogens that can also causemeningitis. Typically, these infections are treated with antibiotics. Inthe past three decades, there has been a dramatic worldwide increase inantibiotic resistance in OM pathogens. This has resulted in a reductionof the number of effective antibiotics for this disease and begun topose a major public health threat. Thus, the use of antibiotics isbecoming more complicated as resistance increases, necessitating thetesting of microbes before treatment, which can sometimes fatally delaythe necessary treatment. In addition, wide antibiotic use is furthercontributing to the problem of resistance. The need to identify newantibiotics is causing the price of these substances to be so high as tobe prohibitive in some cases. Therefore, there is a need for new,innovative, and cost-effective approaches to prevent and manage thesediseases. It is believed that the defenses of the Eustachian tube (Etube) and the middle ear (tubotympanum) help to maintain the sterilityof the middle ear under normal conditions. To this end, an understandingof the mechanisms that protect the tubotympanum (the middle ear andEustachian tube) from invading organisms and determine the role thatthey play in the pathogenesis of OM could prove useful in identifying anew treatment.

SUMMARY OF THE INVENTION

The pharmaceutical composition and a method of treatment of infectiousdiseases, such as otitis media, paranasal sinusitis, labyrinthitis andmeningitis are described. The composition comprises EP2E or homologuesthereof.

In one preferred embodiment, a method for the treatment of microbialinfections in a mammal is contemplated, which comprises administering tothe mammal a pharmaceutical composition that includes at least onecomponent selected from lactoferrins, lysozyme, and defensins in anamount effective for the treatment of such microbial infections. Themicrobial infections which can be treated by the method of the presentinvention include otitis media, paranasal sinusitis, labyrinthitis andmeningitis. For example, the otitis media, caused by NTHi strain 12, M.catarrhalis, S. pneumoniae serotype 3, and S. pneumoniae serotype 6B.

In one preferred embodiment, the defensins in the composition of thepresent invention are alpha-defensins. In yet another preferredembodiment the defensins are beta-defensins, such as beta-defensin 1,beta-defensin 2 and EP2E. Preferably, the EP2E polypeptide is purifiedfrom a natural source or is synthesized chemically and refolded byoxidative refolding.

In one preferred embodiment, the pharmaceutical composition of thepresent invention is administered orally. In another preferredembodiment, the pharmaceutical composition is administered intranasally.In yet another preferred embodiment, the pharmaceutical composition isadministered by aerosolization or into the ear canal.

The method and the composition of the present invention can be used inany mammal, including, but not limited to a dog, a cat, a horse, aferret, a mouse, a rat, a cow, and a primate. Preferably, such primateis human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gel overlay assay for the anti-microbial activity of ratmiddle ear effusion proteins resolved by acid-urea gel electrophoresis.Lane 1, 0.5 mg protegrin; Lane 2, 1 mg human milk lysozyme and humanbeta-defensin 2; Lane 3, PBS wash of normal middle ear; Lane 4, effusionfrom middle ear treated with 1.0 mg S. typhimurium lipopolysaccharide(LPS).

FIG. 2 shows the fold change of mRNA levels for human lysozyme,lactoferrin, beta-defensin 1 (HBD1) (SEQ ID NO: 39, GenBank AccessionNo. AAC51728) and beta-defensin 2 (HBD2) (SEQ ID NO: 40, GenBankAccession No. AAC69554) from real-time PCR analysis of the expression ofthese molecules in normal and inflamed middle ear tissue (black bars).The β-actin gene served as the internal standard. RNAs from one sampleof normal and one sample of inflamed middle ear tissue were reversetranscribed and subjected real-time PCR. The ordinate is log₁₀ of thefold difference of mRNA levels for each antimicrobial molecule betweennormal and inflamed middle ear mucosa samples. The results are based onthe differences of threshold cycle for each molecule in the normal andinflamed samples.

FIG. 3 is a representative radial inhibition assay for the measurementof the activity of innate immune molecules against nontypeableHaemophilus influenzae strain 12 (NTHi strain 12), Moraxella catarrhalisstrain 035E (M. cat), Streptococcus pneumoniae serotype 3 (Sp3) andStreptococcus pneumoniae serotype 6b (Sp 6b). Three doses (4 μg, 10 μgand 40 μg) of human lysozyme (Lz) and lactoferrin (Lf) were testedagainst the bacteria. For human beta-defensin 1 (hBD-1) andbeta-defensin 2 (hBD-2), two doses (4 μg and 10 μg) were tested. Eachdose was delivered in a total volume of 4 μl. The control well receivedonly solvent (0.01% acetic acid). The diameter of the inhibition zonewas measured and averaged in three separate experiments.

FIG. 4 is a graph demonstrating the inhibition of NTHi growth bybeta-defensins 1 and 2, lysozyme and lactoferrin in a colony formationassay.

FIG. 5 shows the effect on the ultrastructure of NHTi after treatmentwith natural antibacterial agents. A) No treatment showing normalappearance of NTHi. An inner membrane encloses the nucleic acidcompartment. The outer surface membrane encloses a compartment filledwith amorphous appearing material. Bar=100 nm. B) Treatment withlysozyme (100 μg/ml). Few completely lysed bacteria were observed.Bar=0.5 micron.

FIG. 6 is an ultra-structural analysis of the effect of humanbeta-defensin 2 and lysozyme on NTHi showing the damage caused to thebacteria by the treatment. Untreated NTHi are shown in panel A. Thebacteria were treated for three hours with β-defensin 2 (10 μg/ml)(panel B), or with 1 mg/ml human milk lysozyme (panel C). Bar=0.2micron.

FIG. 7 is an ultra-structural analysis of the effect of humanbeta-defensin 2 and lysozyme on S. pneumoniae serotype 3 showing thedamage caused to the bacteria by the treatment. Untreated bacteria areshown in panel A. The bacteria were treated for three hours with humanbeta-defensin 2 (10 μg/ml) (panel B), or with 1 mg/ml human milklysozyme (panel C). Untreated NTHi are shown in panel B. Bar=0.5 micron.

FIG. 8 is an ultra-structural analysis of the effect of humanbeta-defensin 2 on S. pneumoniae serotype 6B showing the damage causedto the bacteria by the treatment. Untreated bacteria are shown in panelA. The bacteria were treated for three hours with human beta-defensin 2(10 μg/ml) are shown in panel B. Bar=0.5 micron.

FIG. 9 is an ultra-structural analysis of the effect beta-defensin 2 andlysozyme on M. catarrhalis. Untreated bacteria are shown in panel A.Results of a 1-minute treatment of the bacteria with lysozyme are shownin panel B and those of a 30-minute incubation with beta-defensin 2 areshown in panel C. Bar=0.5 micron.

FIG. 10 shows immunolabeling of archival temporal bone sections showingno change in beta-defensin 1 expression between otitis media (OM) andcontrol. However, human beta-defensin 2 is expressed at high levels inmiddle ear epithelium from OM patients, but not in normal subjects.

FIG. 11. Multiple sequence alignment of human beta-defensin proteinsfrom Human Genome Project (Schutte et al. 2002 PNAS USA 99:2129-33).DEFB1 (SEQ ID NO: 11); DEFB2 (SEQ ID NO: 12); DEFBP1 (SEQ ID NO: 13);DEFB3 (SEQ ID NO: 14); DEFB4 (SEQ ID NO: 15); DEFB5 (SEQ ID NO: 16);DEFB6 (SEQ ID NO: 17); DEFB7 (SEQ ID NO: 18); DEFB8 (SEQ ID NO: 19);DEFB9 (SEQ ID NO: 20); EP2C (SEQ ID NO: 21); SPAG11(EP2E) (SEQ ID NO:22); DEFB31 (SEQ ID NO: 23); Consensus (SEQ ID NO: 38).

FIG. 12. Induction of rBin1b from rat tubotympanum uponLipopolysaccharide challenge.

FIG. 13. Induction of human SPAG11 (EP2E) (SEQ ID NO: 41, GenBankAccession No. AAG21882) from human middle ear epithelial cells HMEEC-1upon otitis media pathogen challenge.

FIG. 14. Quantitative RT PCR analysis for induction of human SPAG11(EP2E) from human middle ear epithelial cells HMEEC-1 upon otitis mediapathogen challenge.

FIG. 15. Immunohistochemical detection of human SPAG11 (EP2E) on humanarchival temporal bone sections from otitis media patient.

FIG. 16. Radial inhibition assay shows effect of rat Bin1b (SEQ ID NO:42, GenBank Accession No. NP_(—)659555) and human SPAG11 (EP2E) upon OMpathogens.

FIG. 17. Liquid broth assay shows the effect of rat Bin1b and humanSPAG11 (EP2E) upon OM pathogen NTHi.

FIG. 18. Ultrastructural change in OM pathogens treated with rat Bin1band human SPAG11 (EP2E).

FIG. 19. Radial inhibition assay shows effect of rat Bin1b and humanSPAG11 (EP2E) upon E. coli ML35.

FIG. 20. Tissue specific expression of Rat Bin1b in the middle and innerear.

FIG. 21. Sequence alignment of mammalian beta-defensins (BD). Thealignment includes the sequences of four human defensins (hBD-1 (SEQ IDNO: 25), hBD-2 (SEQ ID NO: 24), hBD-3 (SEQ ID NO: 28), hBD-4 (SEQ ID NO:29)), six murine defensins (mBD-1 (SEQ ID NO: 30), (SEQ ID NO: 31), (SEQID NO: 32) to mBD-4 (SEQ ID NO: 33), mBD-7 (SEQ ID NO: 26), mBD-8 (SEQID NO: 27)), three bovine defensins (bBD-1 (SEQ ID NO: 35), bBD-2 (SEQID NO: 36), bBD-12 (SEQ ID NO: 37)), and the bovine trachealantimicrobial peptide (bTAP). Strictly conserved amino acid residues arehighlighted by a black box and residues occurring with a frequency of50% are marked by gray boxes. The alignment was generated using theprograms ClustalW (Higgins, D. G. et al. 1992 Comput Appl Biosci8:189-191) and Alscript (Barton, G. J. 1993 Protein Eng 6:37-40).Because of the low sequence similarity, DALI analysis (Holm, L. &Sander, C. 1996 Science 273:595-603) of the three-dimensional structureswas used to allow a correct placement of the gaps in the sequences ofmBD-7 and mBD-8. Elements of secondary structure found in hBD-1, hBD-2,mBD-7, and mBD-8 are schematically indicated below the alignment. Thenumbering scheme refers to the full-length hBD-2 including theamino-terminal signal sequence.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A method and a composition for the treatment of ear and sinus infectionsare disclosed. The method and composition take advantage of the factthat some of the body's innate defenses, lysozyme, lactoferrin and thebeta-defensins, are typically expressed by middle mucosal ear epithelialcells upon infection. The method and composition also take advantage ofthe fact that lysozyme and the beta-defensins can cause damage to andinhibit the growth of nontypeable Haemophilus influenzae strain 12 (NTHistrain 12), Moraxella catarrhalis strain 035E, and Streptococcuspneumoniae serotypes 3, and 6B, three bacteria most commonly found tocause otitis media (OM). The studies herein show that the innate immunesystem may be one of the primary lines of defense against middle earinfections and, thus, future therapies based on these molecules may holdpromise for the treatment of otitis media and sinusitis.

Surface epithelia, including the epithelium of the middle ear, form thefirst barrier against pathogen invasion. Innate immune moleculesproduced by the epithelial cells provide the host with a constitutive orimmediately inducible defense system that is capable of effectivelydealing with the continuous attacks of a variety of pathogens at themucosal epithelial surfaces. Like that of the lung, the epithelium ofthe nasopharyngeal tract is constantly exposed to a multitude ofmicroorganisms. Although the nasopharynx is connected to the middle earcavity via the Eustachian tube, giving pathogens potential access tothis site, under normal conditions, the middle ear of humans andlaboratory animals remains sterile. Furthermore, non-inflamed tubal andmiddle ear mucosa have been shown to contain relatively few immunocytes.These findings suggest that the components of the innate immune systemmay be important in protecting the tubotympanum (middle ear andEustachian tube) and may be playing the role of the first line ofdefense, prior to the activation of adaptive immunity, against otitismedia pathogens.

The pathogenesis of OM is multi-factorial, with risk factors such asupper respiratory viral infection, poor tubal function, delayeddevelopment of the immune system, as well as other environmental andgenetic factors, play an important role, depending on the age of theinfected individual. Available evidence suggests that the mucociliarysystem, systemic immunity (humoral and cell mediated) and the complementsystem play important roles in the protection of the tubotympanum.Recent evidence, however, suggests that other biological defensesystems—particularly the innate (natural) immune system—may also beplaying a role in the protection of the middle ear against OM pathogens.It is now evident that homeostasis of the nasopharyngeal tract,Eustachian tube and the middle ear is maintained in part byanti-microbial proteins and peptides, including lysozyme, lactoferrin,and the defensins.

In addition, there is a need for non-antibiotic treatment of infectionsin view of the problems associated with decades of antibiotic use andmisuse. The incidence of antibiotic resistance is increasing rapidly tothe point where some microbes are resistant to all of the presentantibiotics known. This requires a careful choice of treatment as wellas reducing the speed of treatment, because it may require testing toidentify which antibiotic will be effective for treating the specificmicrobe. In addition, wide antibiotic use is further contributing to theproblem of resistance. Thus, the need to identify new antibiotics iscausing the price of these substances to be so high as to be prohibitivein some cases.

Thus, one embodiment is a method for the treatment of infection in themiddle ear and sinuses by administering one or more antimicrobialpeptides to the middle ear and/or sinuses to inhibit the growth ofpathogenic microorganisms. This method is based on an antimicrobialmixture for which one or more components are selected from the groupconsisting of defensins, lysozyme and lactoferrin, particularly the betadefensins.

Because molecules of the innate immune system including lysozyme,lactoferrin, and the defensins form the first line of defense of thetubotympanum and paranasal sinuses during otitis media and paranasalsinusitis, these molecules present an option to be used alone againstotitis media and paranasal sinusitis or to be used in combination withother known methods of treatment, such as antibiotics. These innatemediators mount a rapid response against the invading pathogens, beforeadaptive immune mechanisms are mobilized in vivo. As such, thesemolecules represent ideal therapeutic candidates to replace antibioticsas the primary treatment modalities for otitis media, sinusitis, andother types of infections.

The present invention uses these innate immune antibiotic molecules totreat infections. This treatment offers a number of advantages. Unlikeexisting antibiotics, these molecules are unlikely to induce antibioticresistance. A further advantage is that these molecules, because theyare produced by the host, will not induce allergic reactions. Lastly,the method of the present invention is more cost effective then that ofantibiotic treatment.

Lysozyme (Lz)

Lysozyme also known as muramidase, is an important component of innateimmunity against pathogens at mucosal surfaces. It catalyzes thehydrolysis of 1-4-glycosidic bonds between N-acetylmuraminic acid andN-acetyl-D-glucosamine, which are constituents of the cell walls of mostbacteria. Lz has potent antibacterial properties and is thus animportant participant in host defense at mucosal surfaces, pleural fluidand in leukocytes. Recent work has shown that inhibition of cationicantimicrobial proteins such as Lz predisposes the airway epithelium toinfection. At mucosal surfaces, Lz expression is confined to specializedepithelial cells, including those of the serous glands of therespiratory epithelium and the Paneth's cell of the gastrointestinalepithelium. Lz is also produced by macrophages and cells of thegranulocyte lineages.

Chronic middle ear effusions contain high levels of Lz, which isproduced by secretory cells of the middle ear mucosal epithelia, as wellas by PMNs and macrophages. Hence, chronic middle ear effusions ofhumans contain high levels of this protein. The fraction of Lz producedby middle ear epithelial cells, has been shown to account for 50% to 80%of the variation of Lz levels seen in middle ear effusions. The presenceof Lz in serous cells of the tubal glands and the secretory epithelialcells suggests a role for this molecule in the defense of the middle earand Eustachian tube against pathogens. The developmental course of Lzsecretion in the murine tubotympanum suggests that its maturationfollows that of secretory components and that it occurs postnatally.

In the middle ear, lysozyme is produced by the secretory cells of theepithelium, as well as by the PMNs and macrophages. Hence, chronicmiddle ear effusions of humans contain high levels of this protein. Thepresence of lysozyme in serous cells of the tubal glands and thesecretory epithelial cells suggests a role for this molecule in thedefense of the tubotympanum against pathogens.

Lactoferrin (Lf)

Lactoferrin is an iron-binding glycoprotein found in the milk andexocrine secretions of mammals and which is released from neutrophilicgranules during inflammation. Lf receptors have been detected onactivated T and B cells, monocytes, intestinal brush border cells,platelets and neoplastic cells. Ingested microorganisms are destroyed incellular phagosomes containing proteolytic enzymes, including Lf. Arnoldand colleagues demonstrated a bacterial killing property of Lf on avariety of bacteria. It also has been shown that Lf synergisticallyinteracts with immunoglobulins, complement, and neutrophil cationicproteins against Gram-negative bacteria. Moreover, Lf can damage theouter membrane of Gram-negative bacteria. It has been well establishedthat breast milk fed babies are more resistant to otitis media thanbabies fed cow milk. Antibacterial lysozyme and Lf in human milk maycontribute to the protection of breast fed babies from otitis media.

It was recently shown that, in addition to its iron-binding properties,Lf possessed proteolytic activity. This activity was first discoveredwhen strains of NTHi were cultured in the presence of human milk whey.The only substrates identified for the proteolytic activity of Lf todate are two related proteins of NTHi, the non-pilus Hap adhesin and theIgA protease precursor. Other outer membrane proteins of NTHi, such asP2, P5 and P6 are unaffected. The proteolysis of pilus Hap adhesin andthe IgA protease precursor yields several large fragments, suggestingthat Lf attacks specific peptide bonds in these proteins. Furthermore,although the proteolytic activity of Lf causes Hap-positive NTHi strainsto lose their ability to adhere to Chang epithelial cells and removesmost of the IgA protease inhibitor from the outer membrane of thesebacteria, it does not affect the viability of the bacteria. Thebiological importance of the Lf hydrolysis of NTHi is presently unknown,but the effect may be to attenuate the pathogenicity of thismicroorganism.

Lf belongs to the gene family that includes transferrin (Tf) and themelanoma surface antigen P97, and shows a high level of homology in bothits sequence and genomic organization with transferrin. To date, Tfs andLfs from a variety of sources and cells have been cloned. Pierce andcolleagues screened a lactating mammary gland cDNA library with ratliver Tf as probe as well as with a human Lf cDNA in order to pull outthe rat Tf and Lf clones. Their results suggest however, that Lf iseither not expressed by the rat mammary gland or that it is expressed atundetectably low levels. Previous results suggest that a similarsituation may exist in the rat tubotympanum. Previous results hereinwere not able to demonstrate expression of Lf in rat tubotympanalepithelial cells or in the tissue. Without being restricted to thefollowing theory, it is possible that Lf is absent in rat and Tf mayhave replaced its function. Lf has also been detected in middle eareffusion, and has been localized to the serous cell of the Eustachiantube glands and to the cuboidal epithelium area (containing the serouscells) of the transition zone of the middle ear. These data suggest thatLf may play a role against bacterial infection of the middle ear andEustachian tube.

Defensins

Antimicrobial peptides have been identified as key elements in theinnate host defense against infection. The defensins are cationic(polar) molecules with antimicrobial activity and have spatiallyseparated hydrophobic and charged regions which have antimicrobialactivity. In vitro, defensins (at micromolar concentrations) have abroad spectrum of antimicrobial activity against bacteria, fungi, andeven some enveloped viruses. Defensins are considered to be among theearliest developed molecular effectors of innate immunity. They arehighly conserved molecules being present in many animal classes(mammals, birds, insects and amphibians). In mammals and birds,defensins are among the most abundant polypeptides secreted byphagocytic white blood cells involved in host defense against bacteriaand fungi. During phagocytosis, ingested microbes are exposed to veryhigh concentrations of defensins. The defensins may also have roles inprotecting the host, based on their capacity to chemoattract T cells, topromote host immunity, and to activate the classical complement pathway.

Defensins are short peptide molecules with a primary chain length of 29to 35 amino acids and molecular weight of 3.5 to 4.5 kDa. They arecationic, variably arginine rich and contain six conserved cysteineresidues which form three intramolecular disulphide bonds that stabilizea rigid three dimensional K sheet structure. They function as dimers bycreating voltage sensitive channels in the plasma membrane of the targetorganism. The antimicrobial spectrum of defensins encompasses grampositive and gram negative bacteria, fungi and viruses (including HIV &HSV). Defensins may also promote a rapid cellular immune response toinfection via a chemotactic effect on monocytes. In addition to theirantimicrobial actions defensins may accelerate wound healing, by virtueof their mitogenic effect on epithelial cells and fibroblasts.

Based on the pattern of cysteine connectivity, the mammalian defensinscan be divided into two major subgroups termed alpha- andbeta-defensins. In alpha-defensins, invariant disulfide bonds are formedin a 1-6, 24, and 3-5 order (Selsted, M. E. & Harwig, S. S. 1989 J BiolChem 264:4003-4007), whereas beta-defensins exhibit a 1-5, 2-4, and 3-6cysteine pairing (Tang, Y. Q. & Selsted, M. E. 1993 J Biol Chem268:6649-6653). Although disulfide linkages differ, thethree-dimensional structure of both groups of peptides is very similarincluding an antiparallel beta-sheet as a major element of secondarystructure (Zimmermann, G. R. et al. 1995 Biochemistry 34:13663-13671).

In humans alpha-defensins are largely present in neutrophils(alpha-defensins 1-4) and in the small intestinal Paneth cells(alpha-defensins 5 & 6). Beta-defensins have a wider cellulardistribution than alpha-defensins, beta-defensin 1 being expressed bythe pancreas, kidney and respiratory epithelium. Recently beta-defensin2 was demonstrated in the skin and bronchial mucosa.

The first mammalian beta-defensin was discovered from the bovinerespiratory tract, named tracheal antimicrobial peptide (Diamond, G. etal. 1991 PNAS USA 88:3952). Subsequently, lingual antimicrobial peptidewas isolated from the bovine tongue (Schonwetter, B. S. et al. 1995Science 267:1645).

The beta-defensins were originally discovered in mammals asantimicrobial peptides of the airway epithelial cells (Selsted, M. E. etal. 1993 J Biol Chem 268:6641-6648). Four human beta-defensin (hBD)isoforms have been identified to date: hBD-1, -2, -3, and -4 (Bensch, K.W. et al. 1995 FEBS Lett 368:331; Harder, J. et al. 1997 Nature 387:861;Harder, J. et al. 2001 J Biol Chem 276:5707; Garcia, J. R. et al. 2001FASEB J 15:1819). In humans, an abundant beta-defensin 1 (hBD-1) wasinitially discovered by analysis of large quantities of hemofiltrate(Bensch, K. W. et al. 1995 FEBS Lett 368:331-335). hBD-1 mRNA isconstitutively expressed in numerous tissues, including the gingiva,parotid gland, buccal mucosa, and tongue (Mathews, M. et al. 1999 InfectImmun 67:2740-2745; Sahasrabudhe, K. S. et al. 2000 J Dent Res79:1669-1674). A second human beta-defensin (hBD-2) is an inducibleproduct of airway epithelia representing the dynamic component of thelocal epithelial defense system (Schröder, J. M. & Harder, J. 1999 Int JBiochem Cell Biol 31:645-651; O'Neil, D. A. et al. 2000 Infect Immun68:5412-5415). A third human beta-defensin, hBD-3, was isolated fromskin (Harder, J. et al. 2001 J Biol Chem 276:5707-5713). In general,each beta-defensin exhibits a distinct spectrum of antimicrobialactivity. Beta-defensins are either constitutively expressed or inducedupon stimulation by different inflammatory factors. It was reported thatboth hBD-1 and hBD-2 bind and activate the chemokine receptor CCR-6,thus linking innate and adaptive immunity (Yang, D. et al. 1999 Science286:525-528). A sequence alignment of mammalian beta-defensins is shownin FIG. 21.

HE21, identified as one major splicing variant of the human EP2 gene,also contains the specific cysteine motif (Osterhoff, C. et al. 1994Biol Reprod 50:516; Hamil, K. G. et al. 2000 Endocrinology 141:1245;Fröhlich, O. et al. 2001 Biol Reprod 64:1072; Jia, H. P. et al. 2001Gene 263:211).

All hBDs show potent antimicrobial activity, especially againstGram-negative bacteria, whereas the function of HE21 had not beenconfirmed (Harder, J. et al. 1997 Nature 387:861; Harder, J. et al. 2001J Biol Chem 276:5707; Garcia, J. R. et al. 2001 FASEB J 15:1819;Goldman, M. J. et al. 1997 Cell 88:553; Valore, E. V. et al. 1998 J ClinInvest 101:1633; Bals, R et al. 1998 J Clin Invest 102:874).

In mice, mouse beta-defensin (mBD)-1 (SEQ ID NO: 43, GenBank AccessionNo. AAB72003), -2 (SEQ ID NO: 44, GenBank Accession No. CAB42815), -3(SEQ ID NO: 45, GenBank Accession No. AAD29573), -4 (SEQ ID NO: 46,GenBank Accession No. NP_(—)062702), -5 (SEQ ID NO: 47, GenBankAccession No. NP_(—)109659), -6 (SEQ ID NO: 48, GenBank Accession No.NP_(—)473415), -7 (SEQ ID NO: 49, GenBank Accession No. NP_(—)631966),-8 (SEQ ID NO: 50, GenBank Accession No. CAC44635), -9 (SEQ ID NO: 51,GenBank Accession No. NP_(—)631965), -11 (SEQ ID NO: 52, GenBankAccession No. NP_(—)631967), -13 (SEQ ID NO: 53, GenBank Accession No.NP_(—)631969), and -35 (SEQ ID NO: 54, GenBank Accession No.NP_(—)631970) have been identified at the National Center forBiotechnology Information (NCBI) gene bank, although the characteristicsof mBD-5, mBD-9, mBD-11, mBD-13, and mBD-35 have not been published(Huttner, K. M. et al. 1997 FEBS Lett 413:45; Bals, R et al. 1998 InfectImmun 66:1225; Morrison, G. M. et al. 1999 FEBS Lett 442:112; Bals, R.et al. 1999 Infect Immun 67:3542; Jia, H. P. et al. 2000 J Biol Chem275:33314; Yamaguchi, Y. et al. 2001 J Biol Chem 276:315; Bauer, F. etal. 2001 Protein Sci 10:2470). mBD-1 and mBD-3 are regarded as mousehomologs of hBD-1 and hBD-2, respectively, and also showed antimicrobialactivity (Bals, R et al. 1998 Infect Immun 66:1225; Bals, R. et al. 1999Infect Immun 67:3542).

hBD-1 (SEQ ID NO: 39), hBD-2 (SEQ ID NO: 40), and hBD-3 (SEQ ID NO: 55,GenBank Accession No. CAC03097) showed the widespread distribution invarious organs like urogenital tissues, skin, respiratory tracts,intestinal tracts, testis, and placenta (Zhao, C et al. 1996 FEBS Lett396:319; Fulton, C. et al. 1997 Lancet 350:1750; Jr McCray, P. B. et al.1997 Am J Respir Cell Mol Biol 16:343; O'Neil, D. A. et al. 1999 JImmunol 163:6718; Garcia, J. R et al. 2001 Cell Tissue Res 306:257).Although the tissue distribution of mBD-5, mBD-7, mBD-8, mBD-9, mBD-11,mBD-13, and mBD-35 have not been evaluated in mice, the other known mBDisoforms also show the expression in multiple tissues, such as kidney,esophagus, tongue, trachea, and skeletal muscle (Huttner, K. M. et al.1997 FEBS Lett 413:45; Bals, R et al. 1998 Infect Immun 66:1225;Morrison, G. M. et al. 1999 FEBS Lett 442:112; Bals, R. et al. 1999Infect Immun 67:3542; Jia, H. P. et al. 2000 J Biol Chem 275:33314;Yamaguchi, Y. et al. 2001 J Biol Chem 276:315).

A novel antimicrobial peptide Bin1b (SEQ ID NO: 42) was identified inthe rat epididymis and its putative amino acid sequence includes theconserved six-cysteine motif (Li, P. et al. 2001 Science 291:1783).Bin1b is partially homologous with HE21 and more homologous with thechimpanzee epididymal protein EP2E (SEQ ID NO: 56, GenBank Accession No.AAF87722) in its amino acid sequence (Fröhlich, O. et al. 2000 J Androl21:421). Interestingly, Bin1b showed no expression in the other majororgans, such as the lung or kidney.

Subsequently, hBD-4 cDNA was identified and its expression was alsoalmost confined to the testis with much lower expression in the gastricatrium (Garcia, J. R. et al. 2001 FASEB J 15:1819). These two isoformsare unique in their confined expression pattern.

The beta-defensins are mainly produced by epithelial cells of the skin,kidneys, and trachea-bronchial lining of nearly all vertebrates. Becausebeta-defensins are released upon microbial invasion and are located atthe host-environment interface, such as mucosal surfaces and skin, theymay also function to alert the adaptive immune system of vertebrates.Human beta-defensin 2 (hBD-2), encoded by DEFB4 (formerly DEFB2 inhumans) genes (Boniotto, et al. 2003 Genes Immun 4:251-257), produced byepithelial cells, exhibits potent antimicrobial activity againstGram-negative bacteria and Candida, but is not as effective againstGram-positive Staphylococcus aureus. HBD-2 represents the first humandefensin that is produced following stimulation of epithelial cells bycontact with microorganisms such as Pseudomonas aeruginosa or cytokinessuch as TNF-alpha and IL-1 beta. Human beta-defensin 2 functions as anNF-kB target gene in the intestinal epithelium and blocking NF-kBactivation inhibits the up-regulated expression of hBD-2 in response toIL-1 alpha stimulation or bacterial infection. The HBD-2 gene andprotein are locally expressed in keratinocytes associated withinflammatory skin lesions such as psoriasis as well as in the infectedlung epithelia of patients with cystic fibrosis.

HBD-1, encoded by DEFB1 gene, is not affected by IL-1 alpha and otherproinflammatory stimuli, thus suggesting that unlike the inducible HBD-2protein, HBD-1 may serve as a defense in the absence of inflammation.The presence of HBD-1 has been reported in the pars tensa and parsflaccida of the tympanic membrane and in the meatal skin. In situhybridization studies localized the HBD-1 mRNA to the epidermal layer ofthese tissues. The HBD-1 transcripts were also evident in the sebaceousglands and in hair follicles of the meatal skin. In contrast, HBD-1 mRNAwas not detected in the tympanal epithelium of the eardrum.

Human beta-defensin 3 (hBD-3) has recently been isolated from humanpsoriatic scales (Garcia, J. R. C. et al. 2001 Cell Tissue Res306:257-264; Harder, J. et al. 2001 J Biol Chem 276:5707-5713) and iswidely expressed in skin, placenta, tongue, and other oral tissues(Dunsche, A. et al. 2002 Eur J Oral Sci 109:121-124; Garcia, J. R. C. etal. 2001 Cell Tissue Res 306:257-264; Harder, J. et al. 2001 J Biol Chem276:5707-5713; Hong Peng, J. et al. 2001 Gene 263:211-218). hBD-3 isactive against a number of human pathogens, including multiresistantStaphylococcus aureus and vancomycin-resistant Enterococcus faecium(Harder, J. et al. 2001 J Biol Chem 276:5707-5713).

The beta-defensin family has shown activities against gram-positive andgram-negative bacteria, fungi, and enveloped viruses in vitro (Ganz, T.,& R. I. Lehrer 1995 Pharmacol Ther 66:191-205; Singh, P. K et al. 1998PNAS USA 95:14961-14966). The activities of HBD-1, -2, and -4 have beenreported to be predominantly effective against gram-negative bacteriasuch as Escherichia coli and Pseudomonas aeruginosa (Garcia, J. R et al.2001 FASEB J 15:819-1821; Singh, P. K et al. 1998 PNAS USA95:14961-14966), with weak or no activity against gram-positive bacteriasuch as Staphylococcus aureus and Streptococcus pyogenes (Garcia, J. Ret al. 2001 FASEB J 15:819-1821; Harder, J et al. 1997 Nature387:861-862; Schröder, J. M. & J. Harder 1999 Int J Biochem Cell Biol31:645-651). HBD-2 was found to be 10-fold more potent than HBD-1 andexhibited activity against P. aeruginosa at physiological concentrations(100 ng/ml) (Singh, P. K et al. 1998 PNAS USA 95:14961-14966). HBD-4seemed to be less effective than HBD-2 against selected gram-positiveand gram-negative bacteria and yeasts, with the exception of P.aeruginosa, for which HBD-4 displayed a greater antimicrobial activitythan the other known defensin peptides (Ganz, T., & R. I. Lehrer 1995Pharmacol Ther 66:191-205). In contrast, HBD-3 has shown broad-spectrumactivity against both gram-negative and gram-positive bacteria atconcentrations much lower than those for other members of thebeta-defensin family (Harder, J et al. 2001 J Biol Chem 276:5707-5713).In addition, its activity appears to be less salt sensitive than thoseof HBD-1, -2, and -4 (Bals, R et al. 1998 J Clin Investig 102:874-880;Ganz, T., & R. I. Lehrer 1995 Pharmacol Ther 66:191-205; Goldman, M. J.et al. 1997 Cell 88:553-560; Singh, P. K et al. 1998 PNAS USA95:14961-14966). Therefore, HBD-3 is considered the most potentbeta-defensin peptide described thus far.

Defensins are normally sequestered in cytoplasmic granules with theirprimary site of action in phagolysosomes, although some peptide isreleased into the circulation during the course of infection orinflammation. Developmental regulation of the alpha defensins(cryptidins) has been studied in mice, where it has been shown thatcryptidin-6 is the most abundant enteric defensin mRNA in the newborn.Paneth cell mRNAs, including cryptidins-4 and -5, lysozyme, matrilysin,and defensin-related sequences, also were detected in RNA from P1 mouseintestine. Unlike adult mice, where only Paneth cells are immunopositivefor cryptidin, cryptidin-containing cells were distributed throughoutthe newborn intestinal epithelium and not in association withrudimentary crypts. In studies of human newborns, it was found thattheir neutrophils were not deficient in defensins although they weredeficient in bactericidal/permeability-increasing protein (BPI), a 55 kDpolypeptide that binds with high affinity to bacteriallipopolysaccharides and kills gram-negative bacteria. The human entericdefensins, on the other hand, are present at sub-adult levels in thedeveloping embryo and it is postulated that levels of enteric defensinexpression in the fetus may be characteristic of an immaturity of localdefense, which is thought to predispose infants born prematurely toinfection from intestinal microorganisms.

In vitro, the defensins (at micromolar concentrations) have a broadspectrum of antimicrobial activity against bacteria, fungi, and evensome enveloped viruses. In mammals and birds, defensins are among themost abundant polypeptides secreted by phagocytic leukocytes andepithelial cells involved in host defense. During phagocytosis, ingestedmicrobes are exposed to very high concentrations of defensins. Thedefensins may also have roles in protecting the host, by their capacityto chemoattract T cells, to promote host immunity, and to activate theclassical complement pathway.

Surface epithelia, including the epithelium of the middle ear, form thefirst barrier against pathogen invasion. Innate immune moleculesproduced by the epithelial cells provide the host with a constitutive orimmediately inducible defense system that is capable of effectivelydealing with the continuous attacks of a variety of pathogens at themucosal epithelial surfaces. Like that of the lung, the epithelium ofthe nasopharyngeal tract is constantly exposed to a multitude ofmicroorganisms. Although the nasopharynx is connected to the middle earcavity via the Eustachian tube, giving pathogens potential access tothis site, under normal conditions, the middle ear of humans andlaboratory animals remains sterile. Furthermore, non-inflamed tubal andmiddle ear mucosa have been shown to contain relatively few immunocytes.These findings suggest that the components of the innate immune systemmay be important in protecting the tubotympanum (middle ear andEustachian tube) and may be playing the role of the first line ofdefense, prior to the activation of adaptive immunity, against otitismedia pathogens.

Active Variants and Homologues

Lactoferrin, lysozyme, and the defensins are well-known molecules whichhave previously been analyzed in detail. Thus, the regions necessary foractivity of these molecules are easily identified. Active variants whichpossess truncations, single or multiple nucleotide transitions andtransversions, and additions may be easily identified and produced withlittle to no loss of activity. In one embodiment, the active variantswhich are produced contain at least about 60% of the activity of thenon-mutagenized protein, including, but not limited to: 65%, 70%, 75%,80%, 85%, 90%, 95%, and 99% of the activity.

For example, any mammalian lysozyme may be cloned using methods known toone of skill in the art, for example, by PCR or by isolating orpurchasing a full-length cDNA from a library (also see Kikuchi, et al.U.S. Pat. No. 4,945,051, issued Jun. 24, 1987 for a method of producingrecombinant human lysozyme—both of which are herein incorporated byreference). Using the information in the literature which identifiesregions necessary for lysozyme activity, variants can be produced bydeleting, truncating, or mutating regions which are not necessary. Forexample, Connely, et al. U.S. Pat. No. 6,111,081, issued May 30, 1997and U.S. Pat. No. 5,571,691, issued Oct. 28, 1993 (both hereinincorporated by reference) provide a method for cloning and mutagenizinglactoferrin, without affecting its iron binding activity. A number ofvariants are disclosed.

The beta-defensins are peptides which are generally between about 38 and42 amino acids which make up a chain having a net charge of +4 to +10.They are further characterized by their content of half-cysteineresidues which are distributed in the peptide chain separated by severalintervening residues. The first and second half-cysteins are separatedby 6 intervening residues; the second and third half-cysteins areseparated by 3 or 4 intervening residues; the third and fourthhalf-cysteins are separated by 9 intervening residues; the fourth andfifth half-cysteins are separated by 5 or 6 intervening residues; andthe fifth and sixth half-cysteins are adjacent. Furthermore, thecysteine residues are paired via disulfide bonds in a characteristicmanner: the first cysteine to the fifth cysteine; the second cysteine tothe fourth cysteine, and the third cysteine to the sixth cysteine. Somebeta-defensins are characterized by a pyroglutamate residue at the aminoterminus which makes these molecules resistant to most aminopeptidases.Thus, one of skill in the art, having this detailed information aboutbeta-defensins as well as that in the literature could identifymutations which would have the least affect on the antimicrobialactivity (see also Selsted, et al., U.S. Pat. No. 6,211,148, issued Dec.11, 1997 herein incorporated by reference).

However, in all cases, the antimicrobial activity of the mutants may betested in a radial assay, such as that detailed in Example 1 or anyother assay known to one of skill in the art.

Homologues of these molecules may be identified in all mammals. Thus,homologues may be identified which may be used for the treatment ofinfections in other mammals, including, but not limited to: dogs, cats,horses, monkeys, apes, cows, sheep, pigs, and a variety of zoo animals.One of skill in the art may identify homologues by searching knownpublic and private databases. It is envisioned that most homologues havealready been identified. However, if a homologue has not yet beenidentified, one of skill in the art may identify a homologue using knowntechniques.

For example, should one of skill in the art wish to identify therhinoceros lactoferrin homologue, he or she may produce probes orprimers which are specific to conserved regions of the lactoferrinproteins. The skilled artisan may then purchase or produce a rhinoceroscDNA library and, using the probes or primers, isolate the transferrinhomologue. Other methods known to one of skill in the art may also beused to isolate and/or identify homologues.

Anti-Microbial Composition

The proteins or active variants of the anti-microbial compounds hereinmay be purified from a natural source, such as, but not limited to, abody fluid, milk, or neutrophils (see Selsted et al. U.S. Pat. No.6,211,148). Alternatively, they may be expressed recombinantly andpurified by any method known to one of skill in the art. The proteins oractive variants are said to be “substantially free of naturalcontaminants” if preparations which contain them are substantially freeof materials with which these products are normally and naturally found.Active variants may be produced using methods known to those of skill inthe art. However, typically, the genes coding for the proteins arecloned and mutagenesis is performed on the gene which is then expressedand the mutagenized protein isolated. Natural active variants may alsobe purified from a mammal which naturally produces such variants.

Compositions for use in the methods herein may contain one or moreactive proteins or variants selected from the group consisting oflysozyme, lactoferrin, alpha-defensins, and beta-defensins. In oneembodiment, the composition contains only one of these proteins. In afurther embodiment more than one of these proteins is included in thecomposition, including but not limited to two, three, and four.

In one embodiment, other treatments are included in the composition. Theother treatments may be any treatment which may enhance theanti-microbial properties, reduce the side-effects, enhance uptake, andincrease the comfort of the patient. For example, it may be possible toinclude substances which reduce the drying effect on the membranes, orincrease healing of the membranes in the area in which it is to beadministered.

Vectors Expressing Proteins or Active Variants

It can be envisioned that one method of administering the lysozyme,lactoferrin, alpha defensins, and beta defensins uses expression vectorswhich express these proteins. The expression vectors may be targeted tothe tissue or cell which is infected or which is near the infectedcells. The vectors may be any vectors known to one of skill in the artincluding but not limited to: viral vectors, plasmid vectors, and nakedDNA. Expression from these vectors may be constitutive or may be underthe control of a specific promoter, such as a eukaryotic promoter, or aninducible promoter.

One advantage of this method is that for those patients who experiencechronic otitis or sinusitis, the presence of a vector may allow theeffectiveness of the treatment to last for a longer time.

Method of Administration and Dosage

It is envisioned that the antimicrobial mixture can be administered toany type of infection, by injection, topically, or even orally. Themethod will depend on the type of infection being treated. For thetreatment of otitis media and sinusitis, the antimicrobial mixture maybe administered to the ear or the sinuses. The administration may befrom the outer ear to the middle ear, e.g. to a patient whose ear drumis pierced and a grommet inserted. Alternatively, if the infection isotitis externa, the administration may be using ear drops. If theinfection is of the middle ear, the ear drops may contain a substancewhich allows permeabilization of the antimicrobial molecules across theear drum. In a further embodiment, the drug may be administered orallyor intranasally where the antimicrobial mixture will act as anantimicrobial agent. Alternatively, the antimicrobial mixture may beadministered orally, intravenously, intramuscularly, in the tear ducts,or by inhalation.

Substances which may be used to permeabilize the ear drum and allowentry of the antimicrobial molecules may be any substance whichincreases the permeability of membranes, such as those which are used topermeabilize skin in dermatology. Examples of such substances include,but are not limited to dimethylsulfoxide (DMSO), dimethylacetamide,methyldecyl sulfoxide, cotton seed oil, caster oil derivatives, fattyacid esters, glycerol, vesicles, liposomes, silicone vesicles (see U.S.Pat. No. 5,364,633 herein incorporated by reference), anionicsurfactants, preparations such as those in U.S. Pat. No. 5,500,416,herein incorporated by reference.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by the recipient patient. Such an agentis said to be administered in a “therapeutically effective amount” ifthe amount administered is physiologically significant. Alternatively,the amount may be analyzed by the effect. For example if the chosenamount produces a reduction in the number of microbes.

The dosage of the protein components of the antimicrobial mixture to beadministered may vary with the method of administration and the severityof the condition to be treated. In general, however, a dosage of fromabout 0.1 to 100 mg/kg/dose, and more preferably 0.5 to 50 mg/kg/dose ofthe drug administered 1 to 8 times a day by the intranasal route, orfrom 1 to 10 drops of a solution or suspension administered from 1 to 10and preferably 1 to 6 times a day, to each ear. In a further embodiment,from about 0.01 mg/ml to about 100 mg/ml, including, but not limited to0.1 mg/ml, 1 mg/ml, 2, mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 30 mg/ml, 40mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, and 90 mg/ml isadministered to the ear, sinuses, or upper respiratory tract at leastone time per day. Local administration is preferable because it reducesthe chances of unwanted side-effects. However, for systemicadministration, a dose of from about 0.01 mg/ml to about 1 g/ml may beadministered at least one time per day for at least and including oneday.

The composition for administration may additionally include additives,excipients, thickeners, and other substances which allow for moreeffective administration. Examples include oils, emollients, or othersubstances which increase the effectiveness and comfort of ear drops,nasal sprays, and inhalable compositions. This may also includesubstances which enhance the smell or taste.

Additional pharmaceutical methods may be employed to control theduration. Controlled release preparations may be achieved through theuse of polymers to complex or adsorb the composition. Alternatively, itis possible to entrap the composition into microcapsules, vesicles, orcomparable molecules.

The inhibitory effect of salt is not envisioned to be a problem as longas high concentrations of the antimicrobial molecules are administered.However, to minimize the effect, in one embodiment, the pharmaceuticalcomposition is administered in a salt-free vehicle, including but notlimited to: water or acidified water. In a further embodiment, thepharmaceutical preparation is administered with a salt chelator. In afurther embodiment, the pharmaceutical preparation is protected fromsalt by administration within liposomes, microcapsule, or any othermethod known to one of skill in the art.

The expression of lysozyme (LZ), lactoferrin (LF), and the human betadefensins (HBD) was analyzed in the middle ear and tissue in normal anddiseased ears in the Examples below. The effect of these substances onOM pathogens morphologically and as antimicrobials was then tested. Fromthese results a method and composition for the treatment of OM andsinusitis was developed. Selected embodiments of this invention areillustrated in the Examples below.

The examples 1-7 below demonstrate that lysozyme, lactoferrin,beta-defensin 1 and beta-defensin 2 are expressed in middle ear mucosaand that are effective against the tested pathogens. The expression ofbeta-defensin 2, lysozyme and lactoferrin mRNAs were higher in theinflamed tissue, while the levels of beta-defensin 1 showed minimalchange. The PCR results for beta-defensins 1 and 2 were confirmed byimmunohistochemical analysis. Previous results are consistent with thePCR results, showing that beta-defensin 2 protein was expressed at ahigher level in inflamed mucosa, whereas beta-defensin 1 showed nochange.

The bacteria used in the following examples were cultured as follows:Stocks of NTHi12 and M. catarrhalis 035E (generous gift of Dr. Xin-XingGu) were maintained at −80° C. For experiments, the bacteria were platedon chocolate agar and incubated overnight at 37° C. in 5% CO₂. A singlecolony was then picked and transferred to 10 ml of brain heart infusion(BHI, Becton Dickinson, Cockeysville, Md.), supplemented with hemin (10μg/ml, Sigma, St. Louis, Mo.) and NAD (10 μg/ml, Sigma, St. Louis, Mo.),and allowed to grow overnight. In the morning, 1/10 volume of theovernight culture was transferred to fresh medium and incubated for3-hours. The subculture was then washed twice with 10 mM sodiumphosphate and the O.D. at 620 nm was determined. For the S. pneumoniaeserotypes 3 and 6B (generous gift of Dr. Xin-Xing Gu), a 50 μl aliquotof the −80° C. stock was thawed on ice, added to 10 ml of Todd-Hewittbroth (THB, Becton Dickinson, Cockeysville, Md.) and cultured overnight.One tenth of the volume of the overnight culture was then transferred tofresh medium and incubated for 3-hours. The subculture was then washedtwice with 10 mM sodium phosphate and the O.D. at 620 nm was determined.

EXAMPLE 1

Expression of innate immune molecules: Lysozyme, Lactoferrin, and humanbeta defensins in the middle ear and the middle ear mucosa normally andin response to pathogens

As shown in FIG. 1, a gel overlay assay was performed to show thatexperimentally induced effusion in rats contained moleculeselectrophoretically consistent with the molecules of innate immunity andwhich had anti-microbial activity. Rats were inoculated with 1.0 mg ofS. typhimurium endotoxin in 100 μl of sterile saline. Animals weresacrificed after 48 hours and the effusion was collected. Themicrobicidal activity of the samples was then evaluated using the gelover-lay assay. The radial assay method of Lehrer and coworkers wasused, with minor modifications (Lehrer et al., 1991 J Immunol Methods137:167-73). The subcultured bacteria (4×10⁶ CFU/10 ml underlay gel)were mixed with melted underlay gel at 42° C. (0.1 X culture broth—BHIfor NTHi and Moraxella and THB for S. pneumoniae-10 mM sodium phosphate,0.8% low electroendosmosis (EEO)-type agarose) and poured into 8 cm×8 cmsquare Petri plates. The gel was allowed to solidify in the Petri platesand wells were punched out with a calibrated 200 μl micropipette tip,cut off at the 50 μl mark (approximately 3 mm). The antimicrobialpeptides and proteins were dissolved in 0.01% acetic acid and 4 μl wasadded to each well. The plates were then incubated for 3 hours at 37° C.in the 5% CO₂ incubator. They were next overlaid with the overlay gel(0.5× culture broth, 10 mM sodium phosphate, 0.8% low EEO-type agarose)and covered. The plates were then allowed to incubate overnight at 37°C. in a 5% CO₂. The diameter of the zones of inhibition produced by eachof the antimicrobial molecules against the OM pathogens (NTHi, M.catarrhalis and S. pneumoniae) was then measured in three separateexperiments and the average and standard deviation were calculated. Asshown in FIG. 1 lane 4, the areas corresponding to low molecular weightpeptides or proteins (consistent with β-defensins) as well as thosecorresponding to larger proteins (consistent with lysozyme, lactoferrin,SP-A and SP-D) display anti-microbial activity in this assay (FIG. 1).

Next, normal and inflamed human middle ear tissues derived frombiopsies, was analyzed by real-time PCR for lysozyme, lactoferrin,beta-defensin 1 and beta-defensin 2. Real-time PCR was performed asfollows. Human middle ear mucosa was collected from patients undergoingsurgery. Total RNA was isolated by using the TRIzol reagent (Gibco BRL,Rockville, Md.), according to the manufacturer's protocol. cDNA wasgenerated by reverse transcription (RT), using the Superscript II-kit(Gibco BRL, Rockville, Md.), according to the manufacturer's protocol.Real-time PCR was carried out using human lysozyme, lactoferrin,beta-defensin 1, beta-defensin 2 and beta-actin primers. The primersequences are as follows: human lysozyme (1223-1395) (XM_006858), (SEQID NO: 1) 5′-ACT TTT TGT TGG GCA AT-3′(forward), (SEQ ID NO: 2) 5′-AGGCTC ATC TGC CTC AG-3′(reverse); human lactoferrin (947-1282) (AF332168),(SEQ ID NO: 1) 5′-TCT CCG CCA GGC ACA-3′(forward), (SEQ ID NO: 4) 5′-GGAGGC CGA GGA GCA-3′(reverse); human beta-defensin 1 (68-321) (XM_005297),(SEQ ID NO: 5) 5′-CGC CAT GAG AAC TTC CTA CCT T-3′(forward), (SEQ ID NO:6) 5′-AGT TCA TTT CAC TTC TGC GTC ATT T-3′(reverse); human beta-defensin2 (41-336) (XM_005029), (SEQ ID NO: 7) 5′-GGG TCT TGT ATC TCC TCT TCTCGT T-3′(forward), (SEQ ID NO: 8) 5′-TGC GTA TCT TTG GAC ACC ATA GTTT-3′(reverse); human beta-actin (696-964) (BC_002409) (SEQ ID NO: 9)5′-CGG GAA ATC GTG CGT GAC AT-3′(forward), (SEQ ID NO: 10) 5′-GCG TACAGG TCT TTG CGG ATG -3′(reverse).

The PCR mixture contained 200 μM of each primer, 2 μl of template cDNA,and 12.5 μl of SYBR Green master mix (Applied Biosystems, Foster City,Calif.) in a final volume 25 μl of total volume. PCR was carried out ina ABI 7700 Sequence Detection System (Applied Biosystems, Foster City,Calif.) using the following thermal cycling program: 2 minutes at 50°C., 10 minutes at 95° C., followed by 50 cycles of 15 seconds at 95° C.and 1 minute at 60° C. A real-time amplification curve was constructedfor each amplification reaction by relating the fluorescence signalintensity (Rn) to the cycle number. The Rn value corresponded to thevariation in the reporter fluorescence intensity before and after PCR,normalized to the fluorescence of an internal passive reference presentin the buffer solution (6-carboxy-x-rhodamine, a rhodamine derivative).The Ct value (cycle threshold; corresponding to the cycle number atwhich a significant increase in the fluorescence signal was firstdetected) was determined for each reaction and served as the basis forcomparison of the relative amount of molecule-specific cDNA in eachreaction. The Ct for beta-actin was subtracted from the Ct values foreach of the other amplified molecules, in order to correct fordifferences in the amount of starting material. The corrected Ct valuesfor each molecule were compared between the normal and inflamed samplesand the cycle difference CD was used to calculate the difference (2CD)for each molecule between the normal and inflamed samples. Human lungcDNA was used as a positive control and for the negative controls, notemplate was included. PCR products were also analyzed byelectrophoresis on a 2% agarose gel containing ethidium bromide in orderto ensure that only the specific product was amplified. The resultsshowed that the transcripts for lysozyme, lactoferrin and beta-defensin2 were present at elevated levels in the inflamed tissues. Thebeta-actin adjusted threshold cycle differences between normal andinflamed samples for lysozyme, lactoferrin, beta-defensin 1 andbeta-defensin 2, were 3.48, 7.38, -0.89 and 10.16, respectively,corresponding to an increase of 11 and 166, and 1150 fold for lysozyme,lactoferrin, and beta-defensin 2 and a decrease of 0.54 fold forbeta-defensin 1 (FIG. 2 and Table 1). TABLE 1 Threshold cycles for humanlysozyme (hLz), lactoferrin (hLf), beta-defensin 1 (HBD-1) andbeta-defensin 2 (HBD-2), compared between normal and inflamed middle earmucosa. Average Threshold Cycles Inflamed (adjusted to beta- Normalactin) Normal-Inflamed hLz 27.37 23.89 3.48 hLf 30.83 23.45 7.38 HBD-127.91 28.80 −0.89 HBD-2 42.46 32.30 10.16

cDNAs derived from one sample of normal and one sample of inflamedmiddle ear tissue were subjected to real-time PCR for the detection ofthe antimicrobial molecules using SYBER Green I dye on a ABI Prism 7700Sequence Detection system. The average of three real-time PCR runs isgiven above for each cDNA. The threshold cycle for beta-actin was 21.94for normal tissue and 19.72 for inflamed tissue (2.22 cycle difference).In order to control for differences in the amount of starting material,this difference of 2.22 cycles was added to the threshold cycles for theantimicrobial molecules to arrive at the adjusted values.

EXAMPLE 2 The Effect of Lysozyme, Lactoferrin and Human Beta-Defensinson OM Pathogens

The radial assay method of Lehrer and coworkers was used, with minormodifications (Lehrer et al., 1991 J Immunol Methods 137:167-73). Thesubcultured bacteria (4×10⁶ CFU/10 ml underlay gel) were mixed withmelted underlay gel at 42° C. (0.1× culture broth—BHI for NTHi andMoraxella and THB for S. pneumoniae-10 mM sodium phosphate, 0.8% lowelectroendosmosis (EEO)-type agarose) and poured into 8 cm×8 cm squarePetri plates. The gel was allowed to solidify in the Petri plates andwells were punched out with a calibrated 200 μl micropipette tip, cutoff at the 50 μl mark (approximately 3 mm). The antimicrobial peptidesand proteins were dissolved in 0.01% acetic acid and 4 μl was added toeach well. The plates were then incubated for 3 hours at 37° C. in the5% CO₂ incubator. They were next overlaid with the overlay gel (0.5×culture broth, 10 mM sodium phosphate, 0.8% low EEO-type agarose) andcovered. The plates were then allowed to incubate overnight at 37° C. ina 5% CO₂. The diameter of the zones of inhibition produced by each ofthe antimicrobial molecules against the OM pathogens (NTHi, M.catarrhalis and S. pneumoniae) was then measured in three separateexperiments and the average and standard deviation were calculated.

After confirming the expression of the innate immune molecules in themiddle ear mucosa, the bacteriostatic/bactericidal effects of thesemolecules on four OM pathogens, NTHi strain12, M. catarrhalis strain035E, S. pneumoniae serotype 3, and S. pneumoniae serotype 6B wasdetermined. Treatment of the bacteria with lysozyme resulted in a slightbut visible inhibition of the growth of M. catarrhalis and S. pneumoniaeserotype 6B (FIG. 3 and Tables 2 and 3). This effect was seen with 40 μgof peptide per well, equivalent to a concentration of 10 mg/ml.Lysozyme, however, had no effect on NTHi and appeared to enhance thegrowth of S. pneumoniae serotype 3 (FIG. 3 and Table 2). Treatment withbeta-defensin 1 was effective only against M. catarrhalis and only atthe high dose (10 μg—equivalent to 2.5 mg/ml). In contrast beta-defensin2, at both concentrations (4 and 10 μg—equivalent to 1 and 2.5 mg/mlrespectively), significantly inhibited the growth of all four bacterialtypes (FIG. 3 and Tables 2 and 3). M. catarrhalis strain 035E was mostsusceptible to β-defensin 2, followed by NTHi strain12, S. pneumoniaeserotype 3 and S. pneumoniae serotype 6B. Lactoferrin treatment,conversely, not only did not inhibit bacterial growth, but appeared toresult in an enhancement of the growth of S. pneumoniae serotype 3 (10μg—equivalent to 2.5 mg/ml), and S. pneumoniae serotype 6B (10 and 40μg—equivalent to 2.5 and 10 mg/ml respectively) (FIG. 3 and Tables 2 and3). TABLE 2 Measurements of the effect of human lysozyme (hLz),lactoferrin (hLf), beta-defensin 1 (HBD-1) and beta-defensin 2 (HBD-2)on the growth of OM pathogens using the radial inhibition assay. Totalamount of protein/ Bacteria peptide added S. S. (in a total pneu- pneu-Mole- volume M. moniae moniae cule of 4 μl) NTHi catarrhalis 3 6B hLz  4μg — — — — 10 μg — — — — 40 μg —   4 ± 1.7 —   5 ± 1.7 (P) (P) hLf  4 μg— — — — 10 μg E — E — 40 μg E E E E HBD-1  4 μg — — — — 10 μg —  7.7 ±0.6 — — (P) 40 μg ND ND ND ND HBD-2  4 μg  9.7 ± 11.7 ± 0.6 8.7 ±  8.7 ±1.2 2.5 (C) (C) 1.5 (C) (C) 10 μg 12.3 ± 14.7 ± 0.6  10 ± 10.7 ± 1.5 2.9(C) (C) 1.3 (C) (C) 40 μg ND ND ND NDDiameter (mm) of the inhibition zone (average of 3 separateexperiments + SD) caused by the different concentrations of theantimicrobial molecules is given in the column below each pathogen. —:No effect; E: Enhanced growth; C: Complete inhibition; P: Partialinhibition; ND: Not done.

The ability of a few of these innate immune mediators: human milklysozyme (Lz), human beta-defensins (HBD) 1 and 2 and human milklactoferrin (LF) to inhibit the growth of non-typeable Haemophilusinfluenzae (NTHi) at other concentrations was also investigated using adifferent type of assay, a colony formation assay. The molecules weretested separately, as well as in combination, and the effect on NTHigrowth was determined using a colony formation assay. Incubation ofcultures of NTHi with 100 μg/ml human milk lysozyme for 3 hours,resulted in an 80% reduction of the number of viable bacteria (FIG. 4).In previous studies, it was shown that the concentration of lysozyme inmiddle ear effusion can be as high as 3.7 mg/ml and thus theconcentrations used in these studies are well within the physiologicalrange. Incubating the bacteria with 10 μg/ml of either humanbeta-defensin 1 or 2 produced an even more pronounced effect, with closeto a 95% reduction in the number of colonies. Interestingly, incubationof the bacteria with a combination of lysozyme and the P-defensins, withor without lactoferrin (1 mg/ml), did not result in a further decreasein the number of viable bacteria. Although these results are slightlydifferent from those produced by the radial assay, without beingrestricted to the following theory, this may be due to the assayconditions or to the concentration tested. For example, the bacteria maybe more sensitive to the antimicrobials in a liquid assay, such as thecolony forming assay.

EXAMPLE 3 Dose Response Analysis

A dose response analysis is performed for each molecule in order todetermine the concentration range where the best effect is observable aswell as a synergistic effect of different combinations. Thus, variousconcentrations of lactoferrin with various concentrations of defensinsare used as in Example 1 and the concentrations at which maximumeffectiveness of the combination occurs are used in further treatments.

EXAMPLE 4 Effect of NaCl

Physiologically, salt concentrations vary depending on the site.However, typically they can be as high as 150 mM. Thus, the saltsensitivity of the antimicrobial molecules was tested. NaCl was added tothe gel in Example 2 to obtain a final concentration of 100 mM. As shownin Table 3, 100 mM salt completely blocked the growth inhibitory effectof lysozyme, lactoferrin and beta-defensin 1 against all bacteriatested. It also blocked the effect of β-defensin 2 against S. pneumoniaeserotype 3 and S. pneumoniae serotype 6B. High salt concentration,however did not inhibit the activity of β-defensin 2 against NTHi and M.catarrhalis, although it did result in a reduction of this effect. TABLE3 Measurements of the effect of 100 mM NaCl on the inhibition of thegrowth of OM pathogens by human lysozyme (hLz), lactoferrin (hLf),beta-defensin 1 (HBD-1) and beta-defensin 2 (HBD-2), using the radialinhibition assay. Total amount of protein/ Bacteria peptide added S. S.(in a total pneu- pneu- Mole- volume M. moniae moniae cule of 4 μl) NTHicatarrhalis 3 6B hLz  4 μg — — — — 10 μg — — — — 40 μg — — — — hLf  4 μg— — — — 10 μg — — — — 40 μg — — — — HBD-1  4 μg — — — — 10 μg — — — — 40μg ND ND ND ND HBD-2  4 μg 8.8 ± 1.0 9.0 ± 0.7 — — (C) (C) 10 μg 11.3 ±0.3  11.0 ± 0.7  — — (C) (C) 40 μg ND ND ND NDDiameter (mm) of the inhibition zone (average of 3 separate experiments± SD) caused by the different concentrations of the antimicrobialmolecules is given in the column below each pathogen. —: No effect; ND:Not done

EXAMPLE 5 Morphological Changes in Response to the Action ofLactoferrin, Lysozyme, and the Human Defensins.

Molecules of innate immunity can damage the cell wall and membranes ofthe OM microbes. Thus, in order to determine what the effect of thesemolecules was, electron microscopy was performed as follows:

Electron microscopy was performed by treating the bacterial cultureswith antimicrobial protein or peptide for 3 hours, mixing with an equalvolume of 5% buffered glutaraldehyde (pH 7.4) and centrifuging (5000×g,for 20 minutes). The bacterial pellets were left in the fixative at 4°C. for 2 hr, post-fixed in 2% osmium tetroxide, dehydrated in ethanoland embedded in Eponate 12 (Ted Pella, Redding, Calif.). Sections weremounted in coated specimen grids, contrasted with uranyl acetate andlead citrate and examined in a transmission electron microscope (CM120BioTwin, FEI-Philips, Hillsboro, Oreg.) operating at 80 kV. Images wererecorded on photographic film and were subsequently digitized.

In order to determine if inhibition of NTHi growth was due to disruptionof the bacterial cell wall and membranes, ultrastructural analysis wasperformed. As shown in FIG. 5 (3 hour), incubation of NTHi with lysozyme100 μg/ml human milk lysozyme caused sufficient damage to the bacterialmembranes that extrusion of the outer membrane could clearly be seen.Morphological changes were also apparent in bacteria treated for 3 hourswith 10 μg/ml human beta-defensins 1 and 2, or a combination of lysozyme(100 μg/ml), human milk lactoferrin (1 mg/ml). Thus these immunemediators function very well as antibiotics in the above studies.

Ultrastructural changes were analyzed in NTHi after treatment withlysozyme and beta-defensin 2 (SEQ ID NO: 40). Ultrastructural analysisof NTHi, S. pneumoniae serotype 3, S. pneumoniae serotype 6B and M.catarrhalis treated with the antimicrobial molecules revealed thatsignificant changes occurred in the bacteria following exposure tobeta-defensin 2 and lysozyme. The morphology of the untreated NTHi isshown in FIG. 6A. FIGS. 6B and 6C show the morphology of NTHi treatedwith 10 μg/ml beta-defensin 2 for 3 hours, and 1 mg/ml of human milklysozyme for 3 hour, respectively. The results showed that in thepresence of P-defensin 2 and lysozyme, NTHi show blebbing of themembranes with extrusion of the cytoplasmic contents into the blebs(FIGS. 6B and 6C). Moreover, none of the untreated bacteria showed anyevidence of blebbing suggesting that the observed changes were notfixation artifacts. From examination of over 100 EM fields, it can beenvisioned that with either of the antimicrobial molecules, at least 30%of the bacteria showed signs of membrane damage. At the time pointsmeasured, there did not appear to be any ruptured NTHi. A one hourtreatment of the bacteria with β-defensin 2 also showed similar results,suggesting that the effect on the bacteria was occurring relativelyquickly. Furthermore, the morphology and the proportion of affected tonon affected cells were similar in NTHi treated with lysozyme for theone hour, as compared to those treated with this molecule for 3 hours.

Treatment of S. pneumoniae serotype 3 with beta-defensin 2 and lysozymealso resulted in damage to the bacteria, although the former moleculeappears to be much more potent. The untreated bacteria are shown in FIG.7A, while FIGS. 7B and 7C show the effect of a three-hour treatment withbeta-defensin 2 and lysozyme, respectively. As seen in FIG. 7B,treatment with beta-defensin 2 results in disappearance of the capsulein certain regions and the lysis of the bacteria. Although lysozyme hasa similar effect, many fewer bacteria appear to be lysed, consistentwith the results of the radial assay.

Human β-defensin 2 showed activity against S. pneumoniae serotype 9B aswell. As shown in FIG. 8A, these bacteria are capsulated with a distinctcell wall. A three-hour treatment with beta-defensin 2, however resultedin damage to the capsule and condensation of the cytoplasmic material(FIG. 8B). The effect of lysozyme was not tested on this bacterium.

The effect of β-defensin 2 and lysozyme on M. catarrhalis was alsotested. As shown in FIG. 9A, untreated M. catarrhalis have a distinctlobulated capsule. Results of a 1-minute treatment of the bacteria withlysozyme are shown in FIG. 9B and suggest that this treatment can resultin the formation of blebs. Samples from later time points, however,showed no evidence of blebs and, as expected, contained more lysedbacteria. As shown in FIG. 9C, a 30-minute incubation of M. catarrhaliswith beta-defensin 2 resulted in substantial damage to the cells.

Of the four molecules tested in the radial inhibition assay,beta-defensin 2 displayed the highest potency and was effective againstall four pathogens tested. NTHi and M. catarrhalis are both Gramnegative bacteria and the effect of beta-defensin 2 on their viabilityis consistent with other studies. None of the treated NTHi showed anyevidence of the presence of mesosome-like structures such as those seenin S. aureus treated with defensins. It is interesting, however, thattreatment of the NTHi with lysozyme, although effective at theultrastructural level, had no effect on the viability of the bacteria.Similar results were obtained by Shimoda and coworkers, when theyexposed S. aureus to defensins (Shimoda et al., 1995 Infect Immun63:2886-91). The effect of beta-defensin 2 on the viability of a Grampositive bacteria, S. pneumoniae, however, was unexpected and is anunexpected finding.

The observed activity of beta-defensin 1 (SEQ ID NO: 39) against M.catarrhalis is also consistent with previous observations of theantimicrobial activities of this molecule against Gram negativebacteria. None of the other pathogens, including NTHi, were affected bybeta-defensin 1 treatment suggesting that this defensin molecule is lessactive than β-defensin 2 against OM pathogens. As expected, bothmolecules showed salt sensitivity and were inhibited by 100 mM salt

Lysozyme caused no detectable inhibition in the growth of S. pneumoniaeserotype 3 and NTHi, while it showed only minimal activity against M.catarrhalis and S. pneumoniae serotype 6b. Lysozyme is a muramidase thatcleaves the glycan backbone by catalyzing the hydrolysis of1-4-glycosidic bonds between N-acetylmuraminic acid andN-acetyl-D-glucosamine, which are constituents of the cell walls of mostbacteria. Studies of the cell wall of S. pneumoniae, however, have shownthat a very high proportion of the hexosamine units (greater than 80% ofthe glucosamine and 10% of the muramic acid residues) are notN-acetylated, thus rendering the peptidoglycan of these bacteria to beresistant to the hydrolytic action of lysozyme. It is of interest thatthe growth of and S. pneumoniae serotype 6b is slightly inhibited bylysozyme, while that of S. pneumoniae serotype 3 is unaffected. Theresults suggest that even minor changes in the cell wall of bacteria,such as those that may exist between the different serotypes of S.pneumoniae, may have a profound effect on the resistance of the bacteriato this innate immune molecule. Consistent with previous observations,lysozyme also displayed salt sensitivity and its effect was inhibited by100 mM salt.

Lactoferrin is an iron-sequestering glycoprotein that predominates inmucosal secretions. The presence of molecules such as lactoferrin allowsfree extracellular iron to be kept at levels (10⁻¹⁸ M) that do not allowbacterial growth and thus represents a mechanism of resistance tobacterial infections by prevention of colonization of the host bypathogens. Although this function of lactoferrin plays an important rolein the protection of mucosal surfaces, bacteria have evolved mechanismsthat allow them to use lactoferrin's iron-sequestering to their benefit.Thus, unlike the results obtained with lysozyme and the β-defensins,treatment of the four OM pathogens with lactoferrin appeared to have agrowth enhancing effect. All four bacteria tested had a positive growthresponse to lactoferrin, with NTHi strain 12 and S. pneumoniae serotype3 showing a higher sensitivity than the other two. This result is,however, not surprising, as M. catarrhalis has been shown to be able touse lactoferrin as an iron source for growth in vitro and Streptococcuspneumoniae has been shown to specifically recognize and bind humanlactoferrin. Moreover, other bacteria have also been shown to be able touse lactoferrin for growth. When grown under iron starvation, Neisseriameningitidis expresses receptors for transferrin and lactoferrin in theouter membrane. Iron transport mutants of Helicobacter pylori have alsobeen shown to be able grow in the presence of lactoferrin andtransferrin. Lactoferrin saturated with iron has been shown to enhancethe intracellular growth of Legionella pneumophila in HeLa cells.However, it is possible that the effect of lactoferrin in enhancing thegrowth of these microbes may be restricted to the in vitro environmentand when administered in vivo, lactoferrin may exhibit an inhibitoryrole. In addition, not all bacteria have evolved methods to uselactoferrin, thus, lactoferrin may be more useful against bacteria otherthan those currently found most commonly in OM. It is envisioned thatthe bacteria found most commonly in OM may change in the future withother bacteria becoming more predominant as causative agents of OM.Thus, antimicrobials such as lactoferrin may become more useful.

The results of the electron microscope studies suggest thatbeta-defensin 2 and lysozyme have a profound effect on structuralintegrity of NTHi, S. pneumoniae serotype 3, S. pneumoniae serotype 6Band M. catarrhalis. Beta-defensin 2 is a cationic peptide known todamage bacterial outer membranes, while lysozyme is known to disrupt thebacterial cell wall. These findings appear to be consistent with earlierobservations of the effect of these molecules on bacterial integrity.

The following U.S. patents contain related subject matter and are hereinincorporated by reference: U.S. Pat. Nos: 5,242,902; 4,268,519;5,240,909; 5,834,424; 4,961,927; 5,962,410; and 5,910,479.

Thus, in one embodiment of the invention, the pharmaceutical preparationherein comprises β-defensin 2. In a further embodiment, thepharmaceutical preparation herein comprises beta-defensin 2 andlysozyme. In a further embodiment, the pharmaceutical preparation hereincomprises beta-defensin 2, lysozyme, and beta-defensin 1. It isenvisioned that for the treatment of some microbes, lactoferrin is notused due to the fact that these microbes have developed ways of usinglactoferrin. In a further embodiment, a pharmaceutical preparation ofbeta-defensin 2, lysozyme, and beta-defensin 1 is used to treat an OMinfection caused by M. catarrhalis. In a further embodiment, apharmaceutical preparation comprising beta-defensin 2, and lysozyme isused to treat OM caused by S. Pneumonia serotype 6B. In a furtherembodiment, a pharmaceutical preparation of β-defensin 2, lysozyme, andbeta-defensin 1 is used to treat an OM infection caused by NTHi strain12. In a further embodiment, a pharmaceutical preparation ofbeta-defensin 2 is used to treat an OM infection caused by M.catarrhalis. However, it is envisioned that a pharmaceutical preparationcomprising beta-defensin 2, lysozyme, and beta-defensin 1 at differentconcentrations may be useful for the treatment of any OM infection.

EXAMPLE 6 Expression of Changes in Response to the Action ofLactoferrin, Lysozyme, and the Human Defensins

Immunohistochemistry was performed using Celloidin embedded 20 μM thicksections of normal and inflamed mucosa, from archival temporal bonespecimens were obtained from the House Ear Institute's Temporal BoneCollection. Following de-celloidination with ether and absolute ethanol,non-specific binding sites on the slides were blocked using normal goatserum. The sections were then subjected to antigen retrieval (VectorLaboratories, Burlingame, Calif.) and incubated with polyclonalantibodies against human β-defensin 2—1:500 dilution. Signals weredetected by the avidin/biotin complex (ABC) method (VectorLaboratories). As shown in FIGS. 10A and B (lower panel) beta-defensin 2was expressed in tissue from the diseased, but not the normal middleear. These results are consistent with studies of human beta-defensin 2that have shown this molecule to be highly inducible by inflammatorystimuli, with minimal expression in non-challenged tissue or cells. Theexpression of beta-defensin 1 was also examined in these sample, but, asexpected, there was little to no difference in levels of this moleculein normal versus diseased epithelium (FIGS. 10A and B, upper panel).

EXAMPLE 7 Treatment of an OM Infection Caused by M. catarrhalis

A pharmaceutical preparation comprising Lysozyme, beta-defensin 1, andbeta-defensin 2 at a concentration of 10 mg/ml Lysozyme, 10 mg/mlbeta-defensin 1, and 1 mg/ml beta-defensin 2 is produced foradministration to the middle ear of a patient whose ear drum is pierced.A grommet is inserted into the outer ear up to the middle ear and thepharmaceutical preparation is applied. Application is repeated 2× perday for 2 weeks. If the infection returns, the treatment is repeated.

EXAMPLE 8 Human SPAG11 (EP2E) and its Rat Homolog Bin1b are InducibleBeta-Defensin Peptides with Limited Tissue Expression and AntimicrobialActivity in Tubotympanum

Beta-defensins are cationic peptides produced by epithelial cells thathave been proposed to be an important component of immune function atmucosal surfaces. Similarities between mammalian beta-defensins maypermit the use of rat models to further define the role of thesepeptides in innate host defense. Rat Bin1b (Li P. Et al. 2001 Science291:1783-5) is a peptide that exhibits homology at the gene level tohuman SPAG11 (EP2E), which has homology with beta-defensin familyidentified in man (FIG. 11). We hypothesized that: 1) Middle ear cavityis protected by a highly effective innate immune system consisting ofepithelial cell derived bactericidal molecules as well as complement andphagocytes; and 2) Members of beta-defensins family are expressed by thetubotympanal epithelial cells and are among nature's most potentantimicrobials. Upon these hypotheses, the purpose of this study was todetermine the antimicrobial activity of Bin1b, the tissue distributionof Bin1b expression in the ear, and the effect of bacterial infection onBin1b expression.

Materials and Methods

Q-PCR. Bin1b mRNA expression was assayed by Quantitative polymerasechain reaction (Q-PCR) using cDNA derived from each organ of the ear.Expression of Bin1b was also evaluated in tissues obtained from rat 4 hafter injection to middle ear cavity, which had been sensitized 24 hoursbefore with LPS using Q-PCR.

Peptides, refolding and Antimicrobial Assays. Based on the sequencededuced from Bin1b cDNA, a 43-amino acid peptide was assembled usingautomated [n-(9-fluorenyl)methoxycarbonyl] solid-phase synthesis. Bin1band SPAG11 (EP2E), each of which was homogeneous by electrophoretic andmass spectrum analysis, were quantified by UV absorbance at 280 nm theywere tested for antimicrobial activity against bacterial strains (E.coli ML35p, M. catarrhalis, NTHi, and S. pneumoniae T3) in a CFU assayas described previously (Lee H-Y et al. 2004 BMC Infect Dis. 4:12).Culture densities were measured spectrophotometrically at 600 nm thenresuspended to a final concentration of 10⁶ CFU/ml. An A600 reading of 1corresponds to 1.5×10⁹ CFU/ml for the NTHi, 4.0×10⁸ CFU/ml for M.catarrhalis, 1.0×10⁸ CFU/ml for S. pneumoniae, and 2.5×10⁸ CFU/ml for E.coli. The organisms were incubated with various concentrations ofpeptide at 37° C. with constant shaking for 1 h. Surviving microbes wereplated in triplicate on trypticase soy broth plates (E. coli) orchocolate agar plate (NTHi, M. catarrhalis, and S. pneumoniae).

Immunohistochemistry has been carried out using human temporal bonesections using specific antibody raised against human SPAG11 (EP2E)synthetic peptide.

Results

Rat Bin1b was expressed in the middle ear and E-tube (FIG. 12). In ratmodel, its expression was up regulated upon challenge of bacterialendotoxin LPS. Middle ear showed higher expression than e-tube.Quantitative PCR showed that more than 17,000 mRNA transcripts wereproduced per one million 18S RNA. Human SPAG11 (EP2E) expression wasup-regulated upon challenge of OM pathogens (FIGS. 13 and 14). Thesequence was confirmed from the PCR product.

Specific antibody was raised against synthetic human SPAG11 (EP2E)peptides and was used to detect SPAG11 (EP2E) in human achival temporalbones containing middle ear mucosal layer. SPAG11 (EP2E) peptide wasdetected in epithelial cells of human middle ear in OM patient (FIG.15). Positive signal was found in the OM case, localized in apical andbasal layers of epithelial cells. Effusion also showed positive signal.

Rat Bin1b and human SPAG11 (EP2E) peptides was synthesized chemicallyand refolded by oxidative refolding (Singh, R. R. & Rao, A. G. A. 2002Biochim Biophys Acta 1597:280-291). Synthetic Bin1b inhibited the growthof E. coli, and OM pathogen Moraxella (FIG. 16 and FIG. 19) on radialassay. Their antimicrobial activity was confirmed in solution against OMpathogen NTHi (FIG. 17). Almost complete bactericidal activity was foundin the 2.5-5.0 μg/ml concentration. Their antimicrobial activity wascomparable to rat beta-defensin 2 which is one of potent antimicrobialmolecules. Ultrastructural analysis (FIG. 18) showed that membranestructural changes occurred in the bacteria upon exposure to rat Bin1band human SPAG11 (EP2E). The rat homologue of SPAG11 (EP2E), Bin1b, wasexpressed not only in middle ear and E-tube, but also in the inner earendolymphatic sac (FIG. 20).

Thus, members of defensin family Bin1b in rat and SPAG11 (EP2E) in humanwere expressed in the middle ear and E-tube. Bin1b and SPAG 1I (EP2E)were shown to be inducible peptides with limited tissue expressionduring bacterial infection. SPAG11 (EP2E) expression was up-regulated inOM in human temporal bone. Because it exhibits antimicrobial activityagainst OM pathogen, SPAG11 (EP2E) and its species variants areenvisioned to serve as an innate defense against microbial invasion atspecific mucosal surfaces of tubotympanum in the mammals, including ratand human.

Accordingly, in one preferred embodiment of the present invention, humanSPAG11 (EP2E) synthetic peptide is used as a therapeutic agent againstOM pathogens.

Moreover, innate immune molecules, such as lysozyme, lactoferrin,beta-defensin 1, beta-defensin 2 and EP2E can be used to treat bacterialinfections of the middle ear, sinuses, and meninges and are particularlyeffective against OM pathogens. Resulting in a new, innovative, andcost-effective approach to prevent and treat these diseases.

Although aspects of the present invention have been described in termsof certain preferred embodiments, other embodiments of the inventionwill become apparent to those of skill in the art in view of thedisclosure herein. Thus, obvious changes and modifications may be madewithout departing from the spirit and scope of the invention.Accordingly, the scope of the invention is not intended to be limited bythe foregoing, but rather to be defined only by the claims which follow.

All of the references cited herein are incorporated in their entirety byreference thereto.

1. A pharmaceutical composition comprising a pharmacologically effectiveamount of EP2E polypeptide, wherein said amount of EP2E polypeptide issufficient to treat a microbial infection in a mammal.
 2. Thecomposition of claim 1, wherein said infection is selected from thegroup consisting of otitis media, paranasal sinusitis, labyrinthitis andmeningitis.
 3. The composition of claim 1, wherein said EP2E polypeptideis synthesized chemically and refolded by oxidative refolding.